Method for inhibiting β-protein enzymatic activity

ABSTRACT

A method of inhibiting the function of β-protein, particularly enzymatic activity, such as esterase (cholinesterase) or proteinase activity, is to contact β-protein with a compound which inhibits such enzymatic activity. Examples of such inhibitors are para-amidinophenylmethanesulfonyl fluoride and Ebelactone A, which inhibit the esterase activity of amyloid precursor protein to a greater extent than the esterase activity of acetylcholinesterase.

This application is a continuation-in-part of U.S. Ser. No. 07/819,361,filed Jan. 13, 1992, now U.S. Pat. No. 5,338,663, entitled "Method ofInterfering With Formation of α-Antichymotrypsin-β-Protein Complex,Method of Inhibiting β-Protein Function and Compounds For Use Therein"by Huntington Potter and Usamah Kayyali which is a continuation-in-partof U.S. Ser. No. 07/572,671, filed Aug. 24, 1990, now abandoned,entitled "Method of Interfering With Formation ofα-Antichymotrypsin-β-Protein Complex and Synthetic Peptides For UseTherein" by Huntington Potter (Abandoned). Priority is also claimed toPCT/US93/00325 filed Jan. 13, 1993. The teachings of these applicationsare incorporated by reference. Work described herein was supported byNIH Grants AG08084 and GM35967. The United States Government has certainrights in the invention.

BACKGROUND

Alzheimer's disease is a degenerative disorder of the central nervoussystem that results in a progressive loss of memory and otherintellectual functions, such as reasoning, orientation, and judgement(R. Katzman, Banbury Report 15: Biological Aspects of Alzheimer'sDisease, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,(1983)). Alzheimer's disease occurs in sporadic and familial forms, andin the United States, affects about 600 people for every 100,000. Acharacteristic aspect of the neuropathology of the disease is theoccurrence of proteinaceous deposits referred to as "amyloid" in thecores of brain lesions called neuritic or senile plaques, as well as incerebral blood vessels. The "amyloid" deposits are generally defined as6-10 nm protein filaments with certain staining properties (Abraham, C.R. et al., Cell 52:487-501 (1988)).

Amyloid deposits are also found in the brains of aged humans, althoughnot as extensively as in Alzheimer's disease. Further, Down's syndromepatients more than 30 or 40 years old invariably develop the symptomsand neuropathology characteristic of Alzheimer's disease.

One component of the amyloid deposits was identified as A4 amyloid orβ-protein (β-protein) and is 42 amino acids long (Glenner, G. G and C.G. Wong, Biochem. Biophys. Res. Commun. 120:885-890 (1984)). Thisprotein is apparently derived from a larger membrane-spanning precursorprotein whose RNA is alternately spliced to yield several proteinproducts (Seikoe, D. J., Science 248:1058-1060). These observationssuggested that the amyloid deposits in Alzheimer's disease could resultfrom abnormal expression or posttranslational modification or processingof a normal molecule. Also intriguing was the finding that the geneencoding the amyloid protein precursor is located on chromosome 21,suggesting a common cause for the deposits observed in Down's syndrome,caused by trisomy of chromosome 21, and Alzheimer's disease.

As mentioned above, some cases of Alzheimer's disease appear to befamilial, and are inherited in an autosomal dominant fashion. Linkageanalysis in four families pointed to a lesion on the long arm ofchromosome 21 (St. George-Hyslop, P. H. et al., Science 238:664-660(1987)), which correlated well with the mapping data and similaritiesbetween Down syndrome and Alzheimer disease. Recently, hereditarycerebral hemorrhage with amyloidosis of Dutch origin was reported to belinked to the APP gene, and a point mutation in the coding region of thegene was identified (Van Broeckhoven, C. et al., Science 248:1120-1122(1990); Levy, E. et al., Science 248:1124-1126 (1990)). Patients withthis disease have a form of the β-protein in amyloid deposits inmeningeal and cerebral blood vessels.

However, other studies reported linkage of familial Alzheimer's diseaseto a locus on chromosome 21 distinct from the amyloid precursor protein(APP) gene (Tanzi, R. E. et al., Nature 329:156-157 (1987); VanBroeckhoven, C. et al., Nature 329:153-155 (1987)). Furthermore, therewas no evidence of duplication of the APP gene in cases of familial orsporadic disease. In fact, studies of some families reportedly indicateno linkage to chromosome 21 (Schellenberg, G. D., Science 241:1507-1510(1988). These data suggest that there may be genetic heterogeneity inthe cause of inherited forms of Alzheimer's disease, and other locationsfor the disease gene have been proposed, such as chromosome 14(Weitkamp, L. R., Amer. J. Hum. Genet. 35:443-453 (1983)).

Thus, other components of the proteinaceous deposits in Alzheimer'sdisease may also be of interest and may provide clues to the cause orprogress of the disease. In fact, a second component of the amyloiddeposits has been characterized as α₁ -antichymotrypsin (ACT), which,interestingly, is located on chromosome 14. Abraham et al. reported theidentification of the serine protease inhibitor ACT in amyloid depositsin Alzheimer's disease brain. (Abraham, C. R. et al., Cell 52:487-501(1988).

SUMMARY OF THE INVENTION

This invention relates to a method of interfering with the interactionand/or function of α₁ -antichymotrypsin (ACT) and β-protein and, as aresult, reducing the adverse effects associated with formation of acomplex comprising the two or with activity of the complex components.In one embodiment, the subject invention relates to methods ofinterfering with the formation of a specific complex between ACT andβ-protein and, thus, of reducing adverse effects resulting from complexformation, such as occurs in Alzheimer's disease. In another embodiment,the present invention relates to methods of inhibiting the function ofthe components of the ACT-β-protein complex. In particular, it relatesto methods of inhibiting the function of β-protein, which, as shownherein, has enzymatic activity which might be of physiologicalrelevance.

This invention relates to a novel class of synthetic peptides orpeptide-like compounds which mimic a component of the specific complexwhich forms between the Alzheimer's β-protein and ACT, and which areuseful to interfere with formation of the complex. The invention alsorelates to a method of treating an individual in whom such complexesform, resulting directly or indirectly in an abnormal condition ordisease state, and particularly to a method of treating an individualwith Alzheimer's disease. The synthetic peptides of the presentinvention, which inhibit complex formation between the Alzheimer'sβ-protein and ACT by binding to the β-protein or to ACT, can beadministered to an individual in such a manner as to interfere with theACT-β-protein interaction, and in sufficient quantity so as to have thedesired effect (i.e., reduction of complex formation and the abnormaldisease state).

This invention also relates to inhibitors of the enzymatic or otheractivity of components of the ACT-β-protein complex. In one embodiment,it relates to compositions or compounds which inhibit the activity ofβ-protein, β-protein precursors or β-protein fragments. As describedherein, β-protein precursor proteins and β-protein fragments have beenshown to have esterase activity, including cholinesterase activity (seeExamples 3 and 4), and are being assessed for additional esterase orprotease activities. As a result, the activity of β-protein can beinhibited by administering compositions or compounds which interferewith its cholinesterase activity or other esterase (e.g., lipase) orprotease activity. Such compositions or compounds, which can be knownmaterials or materials designed or developed specifically for inhibitingβ-protein, can be administered to an individual in need of β-proteininhibition by the present method in sufficient quantities to beeffective in inhibiting the β-protein activity.

A further subject of the present invention is a method of identifyingβ-protein inhibitors and inhibitors identified thereby. In the method,β-protein, a substrate upon which it acts (e.g., acetylthiocholine) anda potential β-protein inhibitor are combined under conditions suitablefor β-protein activity; in the presence of a β-protein inhibitor,hydrolysis of the substrate does not occur or occurs to a lesser extentthan would be the case in the absence of the inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the amino acid sequence of Alzheimer amyloid β-protein (SEQ IDNO. 1) (top row) and the active sites of the following five serineproteases: cytotoxic T cell protease (SEQ ID NO. 2); cathepsin G (SEQ IDNO. 3); mast cell protease (SEQ ID NO. 4); trypsin (SEQ ID NO. 5); andchymotrypsin (SEQ ID NO. 6).

FIG. 2 is a graphic representation of the protease-like regulatoryactivity of Alzheimer amyloid β-protein.

FIG. 3 shows the stable interaction of α₁ -antichymotrypsin andβ-protein, demonstrated as described in Example 2.

FIG. 4 is a graphic representation of the time course of hydrolysis ofacetylthiocholine iodide by rat β-peptide 1-40 (5 μg), human β-peptide1-40 (5 μg) and acetylcholinesterase (0.0025 μg).

    ______________________________________                                        Delta A Rat y = 1.9838e-2 + 1.4096e-3x R.sup.Λ 2 = 0.964               Delta A Hum y = 2.5353e-2 + 6.5074e-4x R.sup.Λ 2 = 0.959               Delta A AChE                                                                              y = 2.94853-3 + 2.8559e-3x R.sup.Λ 2                       ______________________________________                                                    = 0.988                                                       

FIG. 5 is a graphic representation of the inhibition bypara-amidinophenylmethanesulfonyl fluoride (pAPMSF) of the hydrolysis ofvarious concentrations of para-nitrophenyl acetate (PNPA) byAPP695-associated esterase isolated from CHO cells. The data for thereaction in the presence of pAPMSF (squares) and in the absence ofpAPMSF (filled diamonds) is presented in the form of a double-reciprocal(Lineweaver-Burk) plot of i/PNPA] (ram⁻¹) versus i/v (wherev=.increment.A₄₀₅ /time). The equations for the upper (filled diamonds)and lower lines (squares), respectively, are:

y=5.4198e-2+0.14479x; R.sup..increment. 2=0.894;

y=4.5422e-2+0.79028x; R.sup..increment. 2=0.947;

FIG. 6 is an illustration of the structure of3,11-dihydroxy-4,6,8,10,12-pentamethyl-9-oxotetradecenoic 1,3-lactone, acompound related to Ebelactone A, and of (p-amidinophenyl)ethanesulfonyl fluoride and (p-amidinoethyl benzyl) methane sulfonylfluoride, two compounds which are related to pAPMSF.

DETAILED DESCRIPTION OF THE INVENTION

The protease inhibitor α₁ -antichymotrypsin (ACT) and the 42-amino acidβ-protein are integral components of the brain amyloid deposits ofAlzheimer's disease, Down's syndrome, and normal aging. This indicatesthat there is a special affinity between ACT and the β-protein, perhapsessential to amyloid formation. A basis for this association issuggested by the similarity of the N-terminus of β-protein to the activesite of serine proteases.

As described herein, in vitro experiments demonstrate that ACT andβ-protein form a complex that reflects the specificity and stability ofa protease-inhibitor interaction. These results suggest a model for theamyloid filament and a physiological function for the β-protein. As alsodescribed herein, β-protein peptides and a β-protein precursor proteinhave been shown to exhibit esterase activity which might havephysiological relevance. It is reasonable to expect that the activity ofβ-protein has a role in the toxic action of the amyloid deposits ofwhich β-protein is a component. It is also reasonable to predict thatthe β-protein has other esterase or protease activity, which can beassessed using known methods, and that that enzymatic activity also hasphysiological relevance.

It has been demonstrated that the β-protein precursor protein termedAPP695 hydrolyzes acetylthiocholine and para-nitrophenyl acetate,indicating that it has esterase activity. In addition, it has been shownthat synthetic Alzheimer β-peptides 1-40 corresponding to rat and humansequences hydrolyze acetylthiocholine in a time-dependent manner andthat the esterase activity is due to an intrinsic property of theβ-peptide, rather than to a non-specific peptide action. As is alsodescribed, both the activity data and radiolabeling data indicate thatthe esterase activity observed is not due to acetylcholinesterase.

Results presented herein make it possible to design or select syntheticpeptides which can be administered to an individual (introduced intocells) in order to interfere with (reduce or prevent) binding of the twocomponents to form the complex. Such synthetic peptides include peptidesand peptide-like compounds (e.g., modified or derivatized peptides)which interfere with interaction of ACT with β-protein, particularlypeptides which "mimic" the active (or binding) site of serine proteases.Such peptides can be short peptides in which the amino acid sequence issufficiently homologous with the sequence of the binding site of aserine protease or with the N-terminus of β-protein as shown in FIG. 1,that they bind with ACT. Alternatively, such peptides can include, inaddition to the sequence sufficiently homologous with the serineprotease binding site or β-protein region, other amino acids (e.g., oneor more amino acids at either or both ends of the binding sitesequence). According to the method of the present invention, a compoundof the present invention is introduced into an individual in such amanner that it interferes with formation of the ACT-β-protein complex.In the present method of interfering with complex formation, a compoundis administered in sufficient quantity and by an appropriate route tohave a therapeutic effect (to result in decreased complex formation).

Results described herein also make it possible to design or selectcompositions or compounds useful for inhibiting the activity of theACT-β-protein complex or its components. Such compositions can be, forexample, agents which inhibit the enzymatic activity of β-protein,β-protein precursor protein or β-protein fragments (referred tocollectively herein as β-protein), either directly or indirectly. Theycan be, for example, esterase inhibitors, particularly cholinesteraseinhibitors, or protease inhibitors which are known agents or aredesigned for the purpose of inhibiting β-protein. Such inhibitors canact directly (e.g., by destroying, binding or otherwise tieing up theβ-protein) or indirectly (e.g., by acting like a "decoy" substrate forthe β-protein activity and, thus, protecting the physiological substrateupon which the β-protein acts). According to the method of the presentinvention, an inhibitor of the activity of a complex component (e.g., aninhibitor of β-protein enzymatic activity) is introduced into anindividual in such a manner and in sufficient quantity to inhibit theactivity and reduce (totally or partially) the adverse effects whichwould otherwise occur.

The following is a description of the demonstration that ACT andβ-protein interact with specificity to form a stable complex in vitroand of the assessment of esterase activity of β-protein peptides andβ-protein precursor proteins, of compounds which can be used tointerfere with the interaction and of inhibitors of the activity ofACT-β-protein complex components, such as β-protein esterase activity.As used herein, the term β-protein includes β-protein (as described onp. 2 above), β-protein precursor proteins and β-protein fragments,present in a complex with α₁ -antichymotrypsin or in an unbound form(not in a complex with α₁ -antichymotrypsin). Identification of suchcompounds and the method by which they are administered are alsodescribed.

The present invention can be used in the treatment of individuals, suchas those with Alzheimer's disease, in whom complex formation wouldotherwise occur or in whom β-protein activity would otherwise have adeleterious effect, such as excessive inactivation or destruction ofacetylcholine or destruction of key proteins in surrounding tissues.

Demonstration of Formation of ACT-β-Protein complex

The extracellular amyloid filaments found in the plaques and bloodvessels of Alzheimer's disease, Down's syndrome, and normal aging,contain two proteins, both intimately associated with the filaments--the42 amino acid β-protein and the serine protease inhibitor, α₁-antichymotrypsin (Glenner, G. G. and C. W. Wong, Biochem. Biophys. Res.Comm. 122:885 (1984); C. L. Masters, et al., EMBO J. 4:2757 (1985); D.J. Seikoe, et al., J. Neurochem. 46:1820 (1986); D. J. Selkoe, et al.,Science 235:873 (1987); Abraham, C. R. and H. Potter, Bio/technology7:147 (1989); D. J. Seikoe, Ann. Rev. Neurosci. 12:493 (1989); B.Muller-Hill, Ann. Rev. Biochem. 58:287 (1989); Neve, R. L. and H.Potter, in Molecular Genetic Approaches to NeuropSychiatric Disease, J.Brosius and R. Fremeau, Eds. (Academic Press, San Diego, in press); C.R. Abraham, et al., Cell, 52:487 (1988); C. R. Abraham, et al.,Neuroscience 32:715 (1989)). Recently, it was found that the majorcomponent of the vascular amyloid in the Dutch variant of hereditarycerebral hemorrhage with amyloidosis (HCHWA-D) is the β-protein, notcystatin C, as in the Icelandic version (HCHWA-I), although theangiopathy appears similar (J. Ghiso, et al., Proc. Natl. Acad. Sci.83:2974 (1986); S. G. van Duinen, et al., Proc. Natl. Acad. Sci. 84:5991(1987)). When brain sections from individuals with this disease wereanalyzed by immunolabeling, ACT was also found to be present (M. M.Picken, et al., Am. J. Pathol. 134:749 (1989)). In contrast, amyloiddeposits found in other diseases do not contain either β-protein or ACT.The biochemical characteristics of ACT and the β-protein suggest a basisfor this special association. First, ACT is a serine protease inhibitorthat functions by acting as a pseudosubstrate and binding covalently toits target protease to form a long-lived complex (J. Travis, et al.,Biochemistry 17:5651 (1978)). Second, an inspection of the sequence ofthe β-protein reveals a region near the N-terminus which shows astriking homology to one segment of the active site of serine proteases,including the key serine amino acid (FIG. 1) (E. Roberts, Neurobiol.Aging 7:561 (1986); Travis, J. and G. S. Salvesen, Ann. Rev. Biochem52.:655 (1983); G. Salvesen, et al., Biochemistry 26:2289 (1987)). Thus,it seemed possible that ACT and β-protein might be able to form acomplex by virtue of a protease inhibitor-like interaction and that thiscomplex contributes to the stability of Alzheimer amyloid filaments.This hypothesis was tested, as described below. Results showed that, infact, the β-protein is able to specifically bind stably to theinhibitory active site of ACT.

As described in Example 1, various synthetic peptides were tested fortheir effect on the inhibition of chymotrypsin by ACT in vitro.

FIG. 2 shows the results of assessment of the ability of ACT to inhibitprotease activity; the assessment was carried out with the syntheticpeptides. As shown in FIG. 2, when the ACT/chymotrypsin molar ratio wasapproximately 1:1, the ACT inhibited over 90 percent of the chymotrypsinactivity. However, when ACT was pre-incubated with an approximatelyfour-fold molar excess of synthetic peptides corresponding to aminoacids 1-12 or 1-28 of the β-peptide prior to the addition ofchymotrypsin, the inhibitory activity of ACT was substantially reducedand the chymotrypsin reaction rate increased 2 to 8-fold. In contrast,pre-incubation with even a 10-fold molar excess of a peptidecorresponding to amino acids 258-277 of the β-protein precursor (whichshows no similarity to the active site of serine proteases), failed tointerfere with ACT. These data indicate that peptides showing similarityto the region around the key serine in the active site of serineproteases (FIG. 1), and in particular the Alzheimer amyloid β-protein,are able to interfere with the inhibitory function of a serine proteaseinhibitor, ACT. The specificity of the interaction indicates that it isoccurring at the inhibitory active site of ACT. The fact that ACT stillshows a substantial ability, even in the presence of the peptide, toinhibit chymotrypsin, probably reflects the fact that the binding of aprotease inhibitor to its target involves many contacts with amino acidsin the full protease active site which the small peptides naturallylack.

Assessment of the Stability and Specificity of Formation of the α₁-ACT-β-Protein Complex

Unlike true substrates that form a transient covalent intermediate withthe protease through the hydroxyl group of its reactive serine, and thenbecome cleaved as the bond breaks and the protease resumes its activestate, protease inhibitors become cleaved, but only very slowly releasetheir attachment to the protease (J. Travis, et al., Biochemistry17:5651 (1978)). Thus, the inhibitors essentially inactivate theprotease in a suicidal, stoichiometric interaction. The fact that serineprotease inhibitors form stable complexes with their target proteasesand that a serine protease inhibitor, ACT, is an integral component ofthe insoluble Alzheimer amyloid deposits, suggests that this proteinmight be incorporated into the filaments through a stableinhibitor-protease interaction. Therefore, the stability of theinteraction between α₁ -antichymotrypsin and the β-protein was assessed.As described in Example 2, radioiodinated peptides corresponding toamino acids 1-12 and 1-28 of the β-protein and the unrelated segment258-277 of the β-protein precursor were prepared, incubated in thepresence of ACT under various conditions, and the mixtureelectrophoresed on SDS polyacrylamide gels. In the absence of ACT, thelow M. W. peptides migrated rapidly at the dye front. However, in thepresence of ACT, a radioactive band was generated at a positioncorresponding to a few thousand M. W. larger than the ACT protein (FIG.3). Thus, the interaction first detected between ACT and the β-proteincan be sufficiently stable to resist denaturation by boiling in SDS andβ- mercaptoethanol. The indication that the ACT-β-protein interactionoccurs at the protease inhibitory site of ACT was confirmed by the factthat the addition of chymotrypsin to ACT prior to the radioactivepeptide prevented the formation of the ACT-peptide complex. Heatdenaturation of ACT prior to the addition of the peptide also preventedcomplex formation.

The specificity of the α₁ -ACT-β-protein interaction was demonstrated byincubating the 1-28 peptide with other proteins (e.g., bovine serumalbumin (BSA), creatine phosphokinase (CPK), carbonic anhydrase (CA),with which it failed to form stable complexes.

In sum, the work described indicates that the two components of theAlzheimer amyloid deposits--ACT and the β-protein--can associate invitro to form an SDS-stable complex. The interaction is specific forboth the peptide and the ACT protein, and likely occurs between theactive protease inhibitor site of ACT and the N-terminus of theβ-protein, which resembles the active site of serine proteases.

These results suggest a model for the structure of the Alzheimer amyloidfilaments. The hydrophobic C-terminal. portion of β-protein moleculesmaking up the filament would be tucked into the interior, while thehydrophilic central segment (amino acids 12-28) would form the surfaceof the filament. The protease active site-related amino acids at theN-terminus would form an arm projecting from the surface of the filamentand available for binding by ACT. However, the latter seems at leastpossible, inasmuch as the β-protein alone can form filaments in vitrohaving a β-pleated sheet conformation (D. A. Kirschner, et al., Proc.Natl. Acad. Sci. 83:503 (1986); E. M. Castano, et al., Biochem. Biophys.Res. Comm. 141:782 (1986); D. A. Kirschner, et al., Proc. Natl. Acad.Sci. 84:6953 (1987)). However, these filaments can easily be solubilizedand therefore must lack a key component or structural conformationcharacteristic of true Alzheimer amyloid filaments. It is possible thatthe binding of a β-protein core filament to the relativelyprotease-resistant ACT provides the required extra stability.

These results also suggest a potential biological function for theβ-protein. Since the β-protein can competitively interact with theactive site of a serine protease inhibitor in vitro, it might beexpected to be able to play a similar role in vivo. By decoying proteaseinhibitors (including the Kunitz-type inhibitor in the β-proteinprecursor), the β-protein, would serve as a proteaseenhancer-effectively increasing proteolytic activity in its vicinity.Overproduction of the β-protein, as would result, for instance, fromincreased proteolytic degradation of the β-protein precursor, could thuslead to a further increase in protease activity, with progressivelyadverse consequences.

Thus, as described herein, it has been shown that, in vitro, a serineprotease inhibitor (ACT) interacts specifically with a serineprotease-like target protein (Alzheimer β-protein) at a region of thelatter which bears striking sequence homology to the active site ofserine proteases, resulting in formation of a stable ACT-β-proteincomplex. The work described herein provides a reasonable molecularmechanism for formation of the insoluble protein filaments that comprisethe amyloid deposits of Alzheimer's disease.

Assessment of Esterase Activity of β-protein, β-protein Fragments andβ-protein Precursor Protein

The β-protein and a β-protein precursor protein have been shown to haveesterase activity, including cholinesterase activity, as describedherein. This presumably results from the protease-like structure of theβ-protein sequence shown in FIG. 1 inasmuch as proteases and esteraseshave a similar mechanism of action and a similar structure in theircatalytic active sites.

The implications of the findings that the β-protein component of theamyloid protein present in Alzheimer's disease has esterase activity aremore evident if one considers two facts about Alzheimer's disease.First, it has been shown that the majority of neurons affected inAlzheimer's disease are cholinergic. This fact has led to theformulation of the cholinergic hypothesis of pathogenesis. Thishypothesis was the background for attempts to alleviate Alzheimer'sdisease symptoms with cholinergic drugs such as acetylcholinesteraseinhibitors. As these drugs proved ineffective, the validity of thecholinergic hypothesis came into question. The other fact is thatseveral researchers have demonstrated that neuritic plaques containacetylcholinesterase activity when examined histochemically. It is nowtempting to infer that the histochemically observed acetylcholinesteraseactivity is due to the activity of the peptides studied in vitro. Thebasis of this hypothesis is the fact that the β-protein is a majorconstituent of neuritic plaques and that true acetylcholinesterase hasnot been isolated from plaques or shown, by immunological assays, to bepresent in the plaques. It is reasonable to expect that thecholinesterase activity exhibited by these peptides might degradeacetylcholine at a faster rate than normal. Such excessive acetylcholineinactivation might compromise cholinergic input into receiving neurons,and result in degeneration. It is well known that neurons, includingcholinergic neurons, when deprived of their afferent innervation,degenerate. Another possibility is that these peptides might also haveprotease activity that could directly destroy key proteins in thesurrounding tissue.

Whatever the normal or pathologic functions of these peptides might turnout to be, designing compounds that selectively modify their observedactivity is one strategy that might prove fruitful in the treatment ofAlzheimer's disease. One explanation for the fact that researchersreport conflicting results about the effectiveness of cholinergictreatment is that the inhibitors used were too non-specific to be usedin a high enough concentration. After all, they were inhibitors of trueacetylcholinesterase, which has many essential functions in the brain.It is quite possible that what is needed are inhibitors that inhibit thepeptide cholinesterase activity but spare the true acetylcholinesterase.It is now possible to characterize the β-protein activity and identifyand/or design inhibitors which act selectively to inhibit the β-proteinwhile not affecting acetylcholinesterase activity. As shown in Example4, inhibitors capable of preferentially inhibiting β-protein activity ascompared with acetylcholinesterase activity have been identified.

Compounds and Methods for Interfering with ACT-β-protein Interactionsand for Interfering with β-protein Esterase Activity

As a result of the discovery of the specific interaction between ACT andAlzheimer β-protein, it is possible to design or select compositions orcompounds which are useful to interfere with (reduce or prevent) thisACT-β-protein interaction. The discovery has also made possible a methodof preventing the interaction in an individual and, thus, a methodpotentially useful for treating or preventing complex formation andadverse effects resulting therefrom. It provides a method of treating orpreventing conditions, such as Alzheimer's disease, Down's syndrome andnormal aging, in which ACT-β-protein complex is formed. It is alsopossible to design or select compounds which inhibit (reduce oreliminate) the esterase activity of the β-protein or otherwise render itinactive or ineffective. In one embodiment, the cholinesterase activityof the β-protein, β-protein precursor or β-protein fragment is inhibitedor inactivated. In this embodiment, it may be desirable to interferewith or inactivate the β-protein cholinesterase activity specifically inan individual and not interfere with other enzymes which have similarbeneficial activities, such as the "true" acetylcholinesterase. Suchcompositions or compounds useful in interfering with ACT-β-proteininteraction or useful in inhibiting the β-protein esterase activity orotherwise rendering it inactive or ineffective; methods of interferingwith ACT-β-protein interaction; methods of inhibiting the β-proteinesterase activity or rendering it inactive or otherwise ineffective; andmethods of administering such compounds or compositions to interferewith ACT-β-protein interaction or to inhibit β-protein esterase activityor render it ineffective are also the subject of the present invention.The compounds and method are particularly useful in reducing formationof ACT-β-protein complexes in individuals in whom such complexes formand result, directly or indirectly, in an abnormal or undesirablecondition or a disease state. For example, such compounds can be used toreduce (totally or partially) or prevent formation of ACT-β-proteincomplexes in individuals who have Alzheimer's disease or would, withoutappropriate treatment, develop Alzheimer's disease.

Compounds of the present invention can be used to interfere with bindingof ACT and Alzheimer's β-protein, either by binding to ACT, thuspreventing formation of the ACT-β-protein complex, or by binding toβ-protein, also preventing formation of the ACT-β-protein complex. Forexample, a peptide corresponding to all or a portion of the amino acidsequence of a serine protease, such as all or a portion of the sequenceof the Alzheimer's β-protein can be used. Alternatively, a syntheticpeptide which mimics the amino acid sequence of the inhibitory activesite of ACT can be used to interfere with ACT-β-protein complexformation. This type of peptide will bind to the β-protein, resulting inthe production of a synthetic peptide-β-protein complex and, in essence,will "tie up" β-protein, precluding the β-protein from interacting withACT. It is also possible to identify compounds which interfere with(reduce or eliminate) ACT-β-protein complex formation and, thus,identify compounds useful for reducing or preventing complex formation.In the method of identifying a compound which interferes with complexformation, ACT, β-protein and a compound to be assessed for its abilityto reduce or prevent complex formation are combined, under conditionssuitable for complex formation. If the compound being assessed is onewhich interferes with ACT-β-protein interaction, complex formation willnot occur or will occur to a lesser extent than would be the case if thecompound were not present. The extent to which complex formation occursis determined using known methods, such as that described in Example 2.Comparison of complex formation in the presence and absence (controlconditions) of the compound being assessed is an indicator of thecompound's ability to interfere (i.e., no difference in complexformation is an indication the compound is not able to interfere).

The peptide compounds of the present invention may have additionaleffects resulting from their influence, direct or indirect, on theprotease inhibitor function of ACT. ACT is a serine protease inhibitorand Alzheimer's β-protein has been shown to inhibit ACT's ability toinhibit a serine protease (i.e., chymotrypsin). Thus, because it decoysprotease inhibitors, β-protein enhances protease activity by effectivelyincreasing proteolytic activity of other molecules (i.e., by reducingACT's ability to inhibit serine proteases).

This view of the interaction between ACT and the β-protein suggestsalternate outcomes from administration of the two types of peptide. Inthe case where the peptide binds to ACT to inhibit complex formation,the peptide may have an inhibitory effect on ACT, resulting inenhancement of protease activity, in a manner similar to that suggestedfor the β-protein itself. In the case in which the synthetic peptidemimics the inhibitory active site of ACT and binds to the β-protein, onemight expect the opposite outcome. That is, the peptide would complexwith the β-protein, preventing its interaction with ACT, and thusblocking the postulated protease enhancing effect of the β-protein. ACTthen would be left free to exercise its function as a proteaseinhibitor.

A synthetic peptide of the present invention can be administered in aphysiologically acceptable carrier (e.g., an appropriate buffer orphysiologic saline solution) to an individual in whom ACT-β-proteincomplex formation is to be interfered with. It can be administered byany route via which it is possible to deliver a therapeuticallyeffective quantity or dose to the individual in a form available to havethe desired effect (reduction of complex formation). For example, asynthetic peptide of the present invention can be administeredparenterally (e.g., intravenously or intramuscularly) in a compositionwhich protects the peptide from degradation. It can be administered withanother agent or drug, such as a drug used conventionally for treatingthe condition being treated or compounds of the present invention whichinhibit β-protein enzymatic activity.

A synthetic peptide of the present invention can be made using knowntechniques, such as recombinant/genetic engineering techniques orchemical synthesis.

Characterization of β-protein activity has been carried out, asdescribed herein, as to its esterase activity, including cholinesteraseactivity. In addition, other esterase activity (e.g., lipase activity)and protease activity of the β-protein can be assessed using knownmethods. For example, para-nitroanlide conjugated substrates designed tocontain various peptide sequences can be synthesized and tested with theenzyme (Sarath, G. et al., "Protease assay methods" In: ProteolyticEnzymes, a Practical Approach, ed R. J. Beynon and J. S. Bond, IRLPress, Eynsham, 1989). Additional information about β-protein activitywill provide guidance as to the type of inhibitors needed (e.g.,esterase inhibitors, protease inhibitors).

Inhibitors can be identified using the β-protein, β-protein precursorprotein and/or β-protein fragments, such as those described herein. In amethod of identifying an appropriate inhibitor, β-protein, a substrateupon which β-protein has been shown to act (e.g., acetylthiocholine,paranitrophenyl acetate (PNPA)) and a potential inhibitor are combined,under conditions suitable for the β-protein to act upon the substrate(e.g., enzymatically to hydrolyze the acetylthiocholine or PNPA). If theagent being assessed is an inhibitor, β-protein activity will be lessthan β-protein activity in the absence of the agent being assessed. Theactivity can be determined using known methods. For example, in the caseof an esterase inhibitor, which reduces the activity of the β-protein,the reduction in activity can be determined by measuring hydrolysis of asuitable substrate, such as PNPA. Similarly, in the case of an esteraseinhibitor, such as a cholinesterase inhibitor, which reduces theactivity of the β-protein, the reduction in activity can be determinedby measuring hydrolysis of the substrate, such as acetylthiocholine.Less hydrolysis of the substrate in the presence of the agent beingassessed than in its absence is an indication that the agent is aninhibitor. Inhibitors may be total or partial inhibitors. If assessmentshows that β-protein has other enzymatic activity (e.g., other esteraseactivity or protease activity), appropriate inhibitors can be identifiedor designed using known methods. It is also possible to interfere withβ-protein activity at the level of the β-protein precursor protein toprevent it from being processed into β-protein (thus, preventing it fromforming an ACT-β-protein complex), in addition or instead of inhibitingits enzymatic activity.

Potential inhibitors can include known compounds such as protease,esterase, lipase, and cholinesterase inhibitors or compounds designed tohave inhibitory activity. Once an inhibitor has been identified, ordesigned, it can be used as identified or designed or can be modified.For example, modifications that alter the partition coefficient,ionization and/or protein binding properties of an inhibitor can becarried out. Modifications of an inhibitor can serve to enhance itsability to (a) enter cells of the brain (cross the blood-brain barrier),(b) resist degradation by cellular enzymes, and/or (c) inhibitβ-protein, for example. Compounds derived by such modifications can haveenhanced inhibitory effect in the central nervous system and enhancedtherapeutic efficacy.

For example, (p-amidinophenyl)methanesulfonyl fluoride (pAPMSF), whichis commercially available and which has been shown herein to inhibitβ-protein esterase activity, can be modified as described above.Candidate analogs include (p-amidinophenyl)ethanesulfonyl fluoride and(p-amidinoethyl benzyl)methanesulfonyl fluoride. These analogs can besynthesized using protocols similar to those described by Laura et al.for making pAPMSF and procedures of Wong and Shaw (Laura, R. et al.,Biochemistry, 19:4859-4864 (1980); Wong, S. C. and Shaw, E., Arch..Biochem. Biophys., 161:536-543 (1974); Wong S. C. and Shaw, E. Arch.Biochem. Biophys., 176:113-118 (1976)).

Ebelactone A has also been shown herein to inhibit β-protein esteraseactivity. Ebelactone A and Ebelactone B are commercially available.Analogs of these compounds, such as those having altered methylation canbe designed. For example,3,11-dihydroxy-4,6,8,10,12-pentamethyl-9-oxo-6-tetra-decenoic acid1,3-lactone can be isolated from actinomycetes using the procedure ofUmezawa et al. (Umezawa et al., J. Antibiotics, 33:1594-1596 (1980)).Alternatively, it may be obtained through chemical synthesis or derivedfrom Ebelactone A by demethylation at carbon 2.

As with compounds useful for interfering with ACT-β-protein complexformation, compounds useful for inhibiting β-protein enzymatic activitycan be administered to an individual in whom β-protein activity is to beinhibited (e.g, an individual with Alzheimer's disease). Such compoundscan be administered in a pharmaceutically acceptable carrier, such as anappropriate buffer or physiological saline solution. They will beadministered by any suitable route by which it is possible to deliver atherapeutically effective quantity or dose to the individual in a formavailable to have the desired effect (inhibition, total or partial, ofβ-protein enzymatic activity, such as esterase or protease activity). Itcan be administered parenterally (e.g., intravenously orintramuscularly). It can be administered with another agent or drug,such as a synthetic peptide of the present invention which interfereswith ACT-β-protein complex formation.

The discovery described herein also makes it possible to identifyACT-β-protein complexes in tissue obtained from an individual, such asin biopsy tissues obtained from an individual suspected of havingabnormally high levels of the complexes. This can be used, for example,to determine the presence or absence and, if desired, the quantity ofACT-β-protein complexes in brain tissue obtained at autopsy.

The present invention will now be illustrated by the following examples,which are not intended to be limiting in any way.

EXAMPLE 1 Assessment of the Protease Regulatory Activity of Alzheimerβ-Protein

Various synthetic peptides were tested for their effect on theinhibition of chymotrypsin by ACT in vitro. The synthetic peptidestested were:

1. a peptide corresponding to amino acids 1-28 of the N-terminal portionof the Alzheimer β protein (See FIG. 1).

2. a peptide corresponding to amino acids 1-12 of the N-terminal portionof the Alzheimer β protein (See FIG. 1, first 12 amino acids); and

3. a control peptide corresponding to amino acids 258-277 of theAlzheimer β-protein precursor, which does not show any similarity to theserine protease active region sequence.

The activity of chymotrypsin was measured by cleavage of the chromogenicsubstrate Succinyl-Ala-Ala-Pro-Phe-nitroanilide. Results showed thatchymotrypsin activity is reduced about 90 percent in the presence of anapproximate equal molar concentration of ACT. If a four-fold molarexcess of either of the two synthetic peptides from the N-terminalportion of the β-protein (corresponding to amino acids 1-28 and 1-12respectively) is added to ACT prior to the protease assay, the peptideinterferes with the inhibitory activity of ACT. In contrast, the controlpeptide corresponding to amino acids 258-277 of the β-protein precursordoes not modulate ACT's ability to inhibit chymotrypsin.

Results are shown in FIG. 2. The data shown for each graph represent theaverage of four independent assays in which 0.5 (left) or 0.6 (right) μgof ACT were incubated for 2 min±peptide in 10 μl 0.1 M phosphate buffer,pH 7, at 20° C. prior to the addition of 0.3 μg of chymotrypsin. After afurther incubation of 2 min, the reaction volume was increased to 0.8ml, 15 μl of 2 mg/ml substrate was added and the reaction followed at OD405 for five minutes. The graphs compare the relative slopes of thereaction curves (all straight lines) normalized to the reaction rate ofchymotrypsin alone. The peptides did not have any independent effect onchymotrypsin.

EXAMPLE 2 Assessment of the Stability of Interaction Betweenα-antichymotrypsin and β-protein

The ability of synthetic fragments of β-protein to inhibit ACT (FIG. 2)indicates that at least a transient complex can form between the twoproteins. The stability of such complexes was demonstrated by incubatingACT with radio-labeled β-protein fragments under various conditions, andthen analyzing the complex formation by polyacrylamide gelelectrophoresis. In some experiments, a protein cross-linking agent(DSS) was used to stabilize the complex prior to gel electrophoresis(FIG. 3, lanes 1-6, 8-11). However, even in the absence ofcross-linking, a complex is formed which is stable to boiling in SDS andβ-mercaptoethanol (FIG. 3, lanes 7, 12 and 13). The addition of an equalmolar amount of chymotrypsin to ACT blocks the active site and preventsthe complex formation (FIG. 3, lanes 5, 10, 11, 14 and 15). Denaturingthe ACT protein by heat (FIG. 3, lane 3) also prevents the complexformation. Immunostaining of the blotted protein with antibodies to α₁-antichymotrypsin (FIG. 3, lower panel) indicated that neither the heatnor the chymotrypsin treatment destroyed the ACT. Thin layerchromatography confirmed that the amount of chymotrypsin added to theACT was not sufficient to digest the peptide.

EXAMPLE 3 Characterization of Esterase Activity of β-protein, β-proteinprecursors and β-protein Fragments

Because of the structural similarities between β-protein and the activesite of serine proteases/esterases, and the finding that ACT binds toβ-protein, experiments were carried out to examine whether theβ-protein, its precursors, or fragments of it exhibited any esterase orprotease activity. The peptides were synthesized to correspond todifferent lengths of amino acids and three well-characterized forms ofamyloid precursor proteins referred to as APP751, APP770 and APP695,were used. They were obtained from Dr. Barry Greenberg (Upjohn,Kalamazoo, Mich.) purified from a bacculovirus expression system. Therelative levels of these three proteins are altered in Alzheimer'sDisease (Neve, R. L. and H. Potter, in Molecular Genetic Approaches toNeuropsychiatric Disease, J. Brosius and R. Fremeau, Eds. (AcademicPress, San Diego). The main protein in the brain is believed to be theAPP695 protein, which differs from APP751 and APP770 in that it lacks adomain with protease/esterase inhibitory function.

Various ester and peptide substrates were tested under differentconditions. When the peptides were incubated with acetylthiocholineiodide or butyrylthiocholine iodide using a modifiedacetylcholinesterase assay (Ellman, G. L. et al., Biochem, Pharmacol7:88-95 (1961), Gorun, V. et al. Anal. Biochem. 86:324-326 (1978)), thefollowing results were observed. The AAP751 and AAP770 proteins(β-protein precursor proteins) did not hydrolyze either substratesignificantly. On the other hand, the AAP695 peptide hydrolyzed bothsubstrates. It had a specific activity of 4-6 nmol/min/mg usingacetylthiocholine as substrate, and 1.8 nmol/min/mg usingbutyrylthiocholine as substrate. A peptide comprised of 1-28 amino acidsof the β-protein had a specific activity of 0.5-1.4 nmol/min/mg withacetylthiocholine versus 1.1 nmol/min/mg with butyrylthiocholine assubstrates. This result indicates that the β protein esterase isdifferent than either acetylcholinesterase or butyrylcholinesterase,each of which strongly prefer their own specific substrates. Thesevalues were consistent in repeated experiments using different batchesof the peptides.

To determine whether the observed activity in APP695 was due to a trueesteratic action rather than a nonspecific protein action, its activitywas compared to that observed with cathepsin G, a serine protease, whichhydrolyzed either acetylthiocholine or butyrylthiocholine onlyminimally, but still favored the butyrylthiocholine toacetylthiocholine.

When the APP695 protein was inhibited with either physostigmine or DFP,it showed 58% and 70% inhibition, respectively of its ability tohydrolyze acetylthiocholine. The inhibitors were added to the AAP695protein 15 minutes prior to the addition of the substrate. The standardassay for esterase activity was then carried out as above. The amount ofinhibitors used was sufficient to completely inhibit a much higheractivity of erythrocyte acetylcholinesterase. The latter findingindicates that the activity observed is not due to an impurity of trueacetylcholinesterase, which would have been otherwise inhibited by theconcentration of inhibitors used. Finally, when the precursors wereelectrophoresed on SDS-PAGE and silver stained, only one major band wasobserved, which is consistent with there being only one major precursorprotein.

One reason this activity has not been reported earlier is thepossibility that a different source of APP might have been used. APPexpressed in a bacterial system would differ from APP expressed in thiseukaryotic system in that the bacterially expressed APP isunglycosylated. More importantly, the assay described and used hereinfor acetylcholinesterase measurement is performed at higherconcentrations of enzyme and is more sensitive than standard techniques.

Experiments using synthetic Alzheimer β-peptides 1-40 corresponding torat and human sequences were performed to measure the time course ofhydrolysis of acetylthiocholine iodide. FIG. 4 shows the resultsobtained. The results indicate that both peptides hydrolyzeacetylthiocholine in a time-dependent manner.

HPLC-pure synthetic 1-40 peptide and 40-1 reverse peptide, obtained fromDr. Bruce Yankner, were used to test whether the esterase activityobserved was due to an intrinsic property of the β-peptide and not to anon-specific peptide action. The peptides can also be produced usingknown methods (e.g., they can be synthesized chemically). The reversepeptide contains exactly the same amino acids as the 1-40 except thatthey are in the reverse order from the carboxy terminal to the aminoterminal. If the esterase action of the peptide were due to non-specificaction, then the arrangement of the amino acids would not be expected toaffect it significantly. However, if it were a true enzyme, then thearrangement of the amino acids is of key importance to its action.Preliminary experiments with these peptides have indicated that the 1-40β-peptide has an activity of 0.26 mU (nmol/min/mg). The reverse peptidehad 1/4 that activity but with a high standard deviation such that it isnot likely to be real. The latter point is confirmed by an inhibitionexperiment in which DFP (a classical serine esterase/protease inhibitor)inhibited the activity of the 1-40 peptide by 75%, but had no effect onthe activity of the reverse 40-1 peptide.

Radiolabeling experiments indicate that the APP695 becomespreferentially labeled with [3H]-DFP as compared to APP751 or APP770.The label is at the position of the major protein band and does notcomigrate with acetylcholinesterase on SDS-PAGE. The significance ofthis finding is that it corroborates the observed results with esteraseactivity: both the activity data and the radiolabeling data indicatethat the esterase enzyme observed is not acetylcholinesterase. Finally,the labeling of the major APP695 band and not of the major bands ofother APP species 751 and 770 argues against the DFP-binding proteinbeing an impurity, particularly since all precursor proteins werepurified in a similar manner.

EXAMPLE 4 Identification of Inhibitors of β-Protein Function

Two inhibitors of the esterase activity of β-protein were identifiedaccording to the present invention. These inhibitors displaypreferential inhibition of β-protein activity as compared withacetylcholinesterase activity.

A) Inhibition of β-Protein Activity by para-Amidinophenylmethanesulfonylfluoride (pAPMSF)

p-nitrophenyl acetate (PNPA) is a substrate for a variety of esterases,including acetylcholinesterases and other esterases. This substrate wasselected as a substrate for β-protein. In particular, amyloid precursorprotein (APP695), purified from Chinese hamster ovary cells, was testedfor its ability to hydrolyze PNPA. APP695 was purified from Chinesehamster ovary cells (CHO cells) essentially as described (Lowery, D. E.et al., J. Biol. Chem., 266: 19842-19850 (1991)). These studiesindicated that the APP695-associated esterase can hydrolyze thissubstrate.

Next, assays were conducted using APP695 and PNPA to identify inhibitorsof the reaction. Para-amidino-phenylmethanesulfonyl fluoride(4-amidinophenylmethanesulfonyl fluoride; pAPMSF; Boehringer Mannheim,Indianapolis, Ind.), a known inhibitor of certain serine proteases wasselected as a potential inhibitor. Aliquots of CHO cell-derived APP695(5 μl containing 0.9 μg) were incubated in the presence or absence ofpara-amidinophenylmethanesulfonyl fluoride (5 mM), in a final volume of20 μl Na/phosphate (20 mM, pH 7.0), for 1 hour at room temperature.Secondly, 20 μl of p-nitrophenyl acetate (PNPA) in phosphate buffer atconcentrations of 1, 2, 4, or 8 mM was added and mixed with the sampleswith or without pAPMSF. The progress of the reactions was monitoredspectrophometrically. Absorbance at 405 nm was read and recorded usingan ELISA plate reader over a period of time. A set of blanks composed ofeach component, minus the protein, was prepared in parallel. For kineticanalyses, the difference between sample and the corresponding blank wasused.

The results of this study indicated that p-amidinophenylmethanesulfonylfluoride (pAPMSF) inhibits the APP695-associated esterase activity by upto 70%. The data were subjected to Lineweaver-Burk analyses (see FIG.5), and the results of this analysis suggest that inhibition iscompetitive.

B) Inhibition of β-Protein Activity by Ebelactone A and Ebelactone B

Ebelactone A and Ebelactone B (CalBiochem, La Jolla, Calif.), which areknown inhibitors of certain esterase, lipase and N-formylmethionineaminopeptidase activities, were selected as potential inhibitors.Ebelactone A and B were tested for their ability to inhibit amyloidprecursor protein-associated esterase activity. Membrane fractionscontaining APP695 were obtained from CH0695 cells essentially asdescribed (Yoshikawa et al., Nature 459: 64-67 (1992)). CH0695 cells areCHO cells which have been transfected with a clone which encodes APP695and which directs the overexpression of APP695. Aliquots (5 μ) ofmembrane fractions from APP695 expressing CHO cells (CH0695) ormock-transfected control cells (TH-8) were incubated in the presence orabsence of each of the following: Ebelactone A (2.6 mM), Ebelactone B(2.5 mM), or DFP (10 mM diisopropyl fluorophosphate; Sigma ChemicalCompany, St. Louis, Mo.) in a final volume of 10 μl Na/Phosphate (20 mM,pH 7.4) for 20 minutes at 37° C. Next, 10 μl of acetylthiocholine iodide(4 mM) in phosphate buffer were mixed with the samples and incubated for85 minutes. At the end of the incubation, 250 μl dithionitrobenzoic acid(DTNB) were added to each sample to stop the reaction and the absorbanceat 405 nm was read and recorded using an ELISA plate reader. The blankswere initially composed of every component minus the correspondingmembrane sample. The corresponding membrane samples were added to theappropriate blanks after stopping the reaction with DTNB. For activitydetermination, the difference between each sample and the correspondingblank was used. The same experiment was performed on purified APPs froma bacculovirus expression system (Lowery et al., J. Biol. Chem.,266:19842-19850 (1991)), except that preincubation with inhibitors wasfor 1 hour, and incubation with substrate was for 55 minutes.

The membrane fractions from CHO cells overexpressing APP695 had anactivity of 6.9 nmol/min/mg versus 2.2 for the TH-8 controls. EbelactoneA did not inhibit the activity of membrane fractions from TH-8 cells,while it completely inhibited the activity of membrane fractions fromCH0695 cells. Ebelactone A also inhibited bovine erythrocyteacetylcholinesterase (ACHE) by 12%. In contrast, Ebelactone B did notinhibit the activity of any of the membrane fractions, while itinhibited AChE activity by 62%. DFP inhibited the activity of membranefractions from control TH-8 cells by 5% and from CH0695 cells by 87%.DFP completely inhibited AChE activity.

Similar results were obtained when APP695 was purified from abacculovirus expression system. In particular, Ebelactone A inhibitedAPP695-associated esterase activity by >90%. In contrast, under theconditions of the experiment, APP695-associated esterase activity wasnot inhibited by Ebelactone B. In addition, under the conditions of theexperiment, no inhibition of APP751-associated esterase activity byEbelactone A was observed, and Ebelactone B gave an abnormally highblank with APP751 such that inhibition could not be determined. Again,AChE activity was more susceptible to inhibition by Ebelactone B (86%)than Ebelactone A (56%). The latter result is consistent with theresults of the first experiment, considering the different times ofpre-incubation with inhibitor (20 minutes versus 60 minutes).

These results suggest that Ebelactone A has specific inhibitory activityagainst APP-associated esterase activity, which is of significance inthe diagnosis, study, and treatment of Alzheimer's Disease.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AspAlaGluPheArgHisA spSerGlyTyrGluValHisHisGlnLys                             151015                                                                        LeuValPhePheAlaGluAspValGlySerAsnLysGlyAlaIleIle                              20 2530                                                                       GlyLeuMetValGlyGlyValValIleAla                                                3540                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AlaAlaPheArgGlyAspSerGlyGly                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        AlaSerPheLysGlyAspSerGlyGly                                                  15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AlaAlaPheMetGlyAspSe rGlyGly                                                  15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AspSerCysGlnGlyAspSerGlyGly                                                   1 5                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       SerSerCysMetGlyAspSerGlyGly                                                   15                                                                        

What is claimed is:
 1. A method of inhibiting enzymatic activity ofAlzheimer amyloid β-protein, comprising contacting Alzheimer amyloidβ-protein with an amount of a compound effective to inhibit enzymaticactivity of Alzheimer amyloid β-protein, wherein the compound isselected from the group consisting of para-amidinophenylmethanesulfonylfluoride and Ebelactone A.
 2. The method of claim 1, wherein theenzymatic activity of Alzheimer amyloid β-protein is an esteraseactivity selected from the group consisting of cholinesterase activityand lipase activity.
 3. The method of claim 2, wherein the compound is acompound which inhibits cholinesterase activity.
 4. The method of claim2 wherein the compound which inhibits esterase activity is a compoundwhich inhibits lipase activity.